CRISPR/Cas9-based knockout reveals that the clock gene timeless is indispensable for regulating circadian behavioral rhythms in Bombyx mori

Circadian rhythms, which are ubiquitous and adaptive, occur across all species, from microbes to humans, in which they organize and modify behavior and physiology. timeless (tim) is a canonical clock gene. The core composition of the Drosophila melanogaster endogenous circadian clock has been extensively investigated; however, in lepidopteran insects, including Bombyx mori, the mechanism is complicated and little is known regarding the participation of tim in the negative feedback loop responsible for behavioral activities. To arrive at a comprehensive understanding of the role of tim in the B. mori endogenous circadian clock, we exploited the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 gene editing system. We attempted to elucidate the functions of tim in the circadian clock of B. mori using Bmtim mutants. The knockouts affected two circadian behavioral activities: adult emergence and embryo hatching rhythms. Quantitative real-time polymerase chain reaction results confirmed that tim-knockouts induced relative reductions in the expression levels, and thereby the oscillation amplitudes, of Bmper and Bmclk messenger RNAs during both the photophase and scotophase. Additionally, the daily rhythmic expression of Bmdbt was upregulated in the photophase and downregulated in the scotophase in a tim-knockout. Our study reveals that tim is integral to the B. mori circadian clock and may be involved in regulating eclosion and hatching rhythms.     

细菌感染对家蚕DNA甲基化与组蛋白乙酰化相关基因表达的影响

【目的】探讨DNA甲基化及组蛋白乙酰化是否参与家蚕 Bombyx mori 免疫反应的调控。【方法】对家蚕与其他生物的DNA甲基转移酶 (DNMT)、组蛋白去乙酰化酶（HDAC）与组蛋白乙酰转移酶（HAT）的蛋白序列进行系统进化分析;利用定量PCR检测家蚕5龄第3天幼虫感染病原菌绿脓杆菌 Pseudomonas aeruginosa 和金黄色葡萄球菌 Staphylococcus aureus 后 BmDNMT 1, BmHDACI-1, BmHDACI-2和 BmHAT 1在家蚕脂肪体组织中的表达变化;给家蚕5龄第2天幼虫注射DNMT, HDAC和HAT抑制剂,观察它们对家蚕感染细菌后的存活率的影响。【结果】系统进化分析显示,BmDNMT1在进化上呈现特殊性,独立于其他昆虫DNMT1的进化,而BmHDACs和BmHAT在进化上相对保守。定量PCR检测表明,在两种细菌感染后,BmDNMT1, BmHDACs 和 BmHAT1 在家蚕幼虫脂肪体中的表达水平均有不同程度的上升。然而,DNMT, HDAC和HAT抑制剂对家蚕幼虫感染细菌后的存活率并无明显影响。【结论】本研究发现感染绿脓杆菌和金黄色葡萄球菌后,家蚕幼虫脂肪体中 BmDNMT1, BmHDACs 和 BmHAT1 的表达水平有不同程度的上调,推测DNA甲基化和组蛋白乙酰化/去乙酰化可能参与家蚕免疫反应的调控。     

Crosstalk between the circadian clock circuitry and the immune system

Various features, components, and functions of the immune system present daily variations. Immunocompetent cell counts and cytokine levels present variations according to the time of day and the sleep-wake cycle. Moreover, different immune cell types, such as macrophages, natural killer cells, and lymphocytes, contain a circadian molecular clockwork. The biological clocks intrinsic to immune cells and lymphoid organs, together with inputs from the central pacemaker of the suprachiasmatic nuclei via humoral and neural pathways, regulate the function of cells of the immune system, including their response to signals and their effector functions. Consequences of this include, for example, the daily variation in the response to an immune challenge (e.g., bacterial endotoxin injection) and the circadian control of allergic reactions. The circadian-immune connection is bidirectional, because in addition to this circadian control of immune functions, immune challenges and immune mediators (e.g., cytokines) were shown to have strong effects on circadian rhythms at the molecular, cellular, and behavioral levels. This tight crosstalk between the circadian and immune systems has wide-ranging implications for disease, as shown by the higher incidence of cancer and the exacerbation of autoimmune symptoms upon circadian disruption. (Author correspondence: g.mazzoccoli@operapadrepio.it)     

细菌感染后家蚕幼虫中肠差异表达基因的鉴定（英文）

作为遭遇各种微生物的前线, 昆虫的消化道在免疫反应中起到重要的作用。为了研究家蚕Bombyx mori肠道免疫反应, 我们利用基于ACP (annealing control primer)的反转录PCR技术, 从中肠组织中鉴定得到18个在经口器感染绿脓杆菌Pseudomonas aeruginosa 和金黄色葡萄球菌Staphylococcus aureus后的差异表达基因,并利用荧光定量PCR分析了其中4个基因在感染后24 h内的动态变化。结果表明： 肽聚糖识别蛋白-L1（PGRP-L1)和一个丝氨酸蛋白酶前体基因特异性地受绿脓杆菌感染后上调; 30kP蛋白酶A基因受绿脓杆菌和金黄色葡萄球菌2种细菌感染后上调。我们的研究鉴定了经口器感染后家蚕中肠中参与入侵细菌识别和免疫信号途径的基因, 可为对这些基因进一步的功能研究提供线索。     

Research progress on the immune mechanism of the silkworm Bombyx mori

The silkworm Bombyx mori L., representing an important economic insect and one of the best models for studying insect immunity, possesses an efficient and sophisticated innate immune system against invasive microorganisms. The innate immune system basically includes humoural immunity and cellular immunity. The humoural immunity, which functions via molecules including humoural factors, lysozymes, phenoloxidase, hemolin, lectins and, in particular, antimicrobial peptides, plays a central role in eliminating the invading pathogens. The cellular immunity is primarily carried out and mediated by plasmatocytes and granular cells of haemocytes in the haemolymph, usually followed by melanization. Additionally, apoptosis, a primary viral defence for insects lacking adaptive immunity, comprises an important part of the silkworm immune system. Currently, there is still the lack of a comprehensive and systematic understanding of the molecular mechanisms of silkworm immunity. We review the latest research progress on silkworm immune mechanisms, including phenoloxidase‐dependent melanization and apoptosis, which is conducive to improving our understanding of the silkworm immune mechanism, clarifying the relationship of various immune mechanisms, and also providing a theoretical basis and reference for the future research of insect immunity.     

The cell adhesion molecule EphA4 is involved in circadian clock functions

Circadian (～24 h) rhythms of cellular network plasticity in the central circadian clock, the suprachiasmatic nucleus (SCN), have been described. The neuronal network in the SCN regulates photic resetting of the circadian clock as well as stability of the circadian system during both entrained and constant conditions. EphA4, a cell adhesion molecule regulating synaptic plasticity by controlling connections of neurons and astrocytes, is expressed in the SCN. To address whether EphA4 plays a role in circadian photoreception and influences the neuronal network of the SCN, we have analyzed circadian wheel‐running behavior of EphA4 knockout (EphA4^?/?) mice under different light conditions and upon photic resetting, as well as their light‐induced protein response in the SCN. EphA4^?/? mice exhibited reduced wheel‐running activity, longer endogenous periods under constant darkness and shorter periods under constant light conditions, suggesting an effect of EphA4 on SCN function. Moreover, EphA4^?/? mice exhibited suppressed phase delays of their wheel‐running activity following a light pulse during the beginning of the subjective night (CT15). Accordingly, light‐induced c‐FOS (FBJ murine osteosarcoma viral oncogene homolog) expression was diminished. Our results suggest a circadian role for EphA4 in the SCN neuronal network, affecting the circadian system and contributing to the circadian response to light.     

家蚕生物钟基因Bmcryl与Bmcry2的克隆及生物信息学分析

隐花色素基因(cryptochrome gene,Cry)是已确认的主要生物钟基因之一,它广泛分布于细菌和真核生物中.昆虫Cry基因分为Cry1,和Cry2两类,果蝇只有Cry1,蜜蜂等膜翅目昆虫只有Cry2.为了研究鳞翅目模式昆虫家蚕Bombyx mori的昼夜生物钟分子调控机制和昆虫CRY蛋白的进化,本研究克隆了家...     

家蚕血淋巴体外黑化观察及相关黑色素合成催化酶基因

观察不同生长期家蚕幼虫血淋巴在体外的黑化速度和对大肠杆菌生长的影响结果显示,随食桑生长幼虫血淋巴的黑化速度逐渐变快,对大肠杆菌生长的抑制作用逐渐增强。RT-PCR实验显示,黑色素合成催化酶Bm Tan、Bm Po-1、Bm Yellow-f和Bm Ddc等的基因在家蚕5 L 3 d血淋巴中表达量高,Bm Black、Bm Yellow和Bm Pah等的基因也有明显表达。q PCR分析显示,黑化病蚕中Bmtan、Bmddc、Bmyellow、Bmebony和Bmblack,尤其Bmddc表达发生了显著上调。与对照相比,Ddc酶的抑制剂能显著抑制脂多糖对血淋巴的诱导黑化作用。用大肠杆菌注射家蚕幼虫,血淋巴中多巴和多巴胺的含量明显上升。这些表明家蚕幼虫血淋巴黑化与防御免疫有关,Bmddc很可能在幼虫血淋巴的免疫黑化中发挥作用。     

家蚕微孢子虫感染对家蚕海龟蛋白(Bmtutl)基因表达水平的影响

【目的】本研究旨在阐明家蚕微孢子虫Nosema bombycis感染不同时间对家蚕Bombyx mori幼虫不同组织中家蚕海龟蛋白(Bombyx Turtle, Bmtutl)基因表达水平的影响,为揭示家蚕微孢子虫的侵染机制奠定基础。【方法】利用生物信息学方法对家蚕海龟蛋白3种亚型Bmtutl-464, Bmtutl-519和Bmtutl-810的序列结构特征进行了分析;利用qPCR检测家蚕微孢子虫感染后12, 24, 48, 72, 96和120 h,家蚕幼虫中肠、血淋巴与脂肪体组织中Bmtutl-464, Bmtutl-519和Bmtutl-810基因表达水平的变化情况。【结果】家蚕海龟蛋白3种亚型的二级结构均主要由无规则卷曲、α螺旋、β转角和延伸链组成,其中无规则卷曲所占比例最高。但是PredictProtein分析发现,Bmtutl-464, Bmtutl-519和Bmtutl-810之间的蛋白/多核苷酸结合位点存在较大差异。qPCR结果表明,感染家蚕微孢子虫后,家蚕幼虫中肠、血淋巴与脂肪体组织中Bmtutl-464, Bmtutl-519和Bmtutl-810基因的整体表达处于被抑制状态,尤其在脂肪体中最为明显：Bmtutl-519和Bmtutl-810基因的表达在家蚕微孢子虫感染家蚕后的72 h开始受到显著抑制,特别是Bmtutl-519基因,其相对表达水平均不到对照的5.0%。【结论】家蚕海龟蛋白这3种亚型的序列结构特征存在较大差异,家蚕微孢子虫感染在一定程度抑制了家蚕幼虫中肠、血淋巴与脂肪体组织中Bmtutl-464, Bmtutl-519和Bmtutl-810基因尤其是Bmtutl-519的表达。结果说明,与其他两种家蚕海龟蛋白亚型相比,Bmtutl-519蛋白可能在家蚕微孢子虫侵染宿主的过程中起主要作用。     

家蚕BmlncR2036调控P25基因启动子活性及参与中肠发育调控

[目的]长链非编码RNA (long non-coding RNA,lncRNA)对家蚕Bombyx mori发育具有重要调控作用.我们在前期研究中发现一个位于家蚕丝素蛋白基因P25附近的lncRNA BmlncR2036.本研究旨在进一步探索BmlncR2036调控家蚕P25基因表达的分子机制.[方法]qPCR检测B...     

家蚕肽聚糖识别蛋白L1参与Imd信号通路

【目的】肽聚糖识别蛋白(peptidoglycan recognition proteins,PGRP)作为一个重要的模式识别受体,在家蚕Bombyx mori的先天免疫中发挥重要的作用。本研究主要探讨家蚕PGRP-L1基因的功能及其所参与的免疫信号通路。【方法】本实验通过RT-PCR扩增获得家蚕PGRP-L1基因。利用微生物诱导实验对5龄起蚕分别注射大肠杆菌Escherichia coli、酿酒酵母Saccharomyces cerevisiae、枯草芽孢杆菌Bacillus subtilis和PBS,12 h后取体壁和头部提取RNA,然后采用RT-PCR和凝胶电泳技术测定BmPGRP-L1基因的表达水平;利用RNA干涉实验向5龄起蚕注射PGRP-L1 dsRNA或PBS,6 h后再分别注射3种灭活的微生物或PBS,然后检测家蚕体壁及头部的免疫相关转录因子(Rel,Cactus和Relish)以及家蚕多个抗菌肽(antimicrobial peptides,AMPs)基因(攻击素基因Attacin,Moricin和葛佬素基因Gloverin)的表达情况。【结果】微生物诱导实验显示,注射大肠杆菌的实验组比注射PBS的对照组Bm PGRP-L1基因转录水平显著上调,而注射酿酒酵母和枯草芽孢杆菌的实验组Bm PGRP-L1转录水平没有变化。利用RNAi技术成功敲低Bm PGRP-1表达,对Bm PGRP-L1敲低的5龄起蚕注射灭活的微生物,敲低实验组relish因子表达低于正常对照组,相应地抗菌肽基因的表达也有不同程度的下调。【结论】结果提示,BmPGRP-L1基因参与家蚕对革兰氏阴性菌大肠杆菌的免疫响应;Bm PGRP-L1基因在家蚕的体壁和头中参与Imd信号通路。     

An immune-induced reeler protein is involved in the Bombyx mori melanization cascade

In this study, we isolated two reeler cDNAs from bacteria-challenged larval fat bodies of the silkworm, Bombyx mori. A reeler domain spanned most of the coding regions of these two cDNAs, and their expression patterns were different in B. mori larval tissues. The reeler1 gene was strongly induced by Escherichia coli K12 and Bacillus subtilis in B. mori larval hemocytes, fat bodies and midguts, but reeler2 was expressed at extremely low levels in these tissues. We focused on the reeler1 gene for functional analysis. Interference by double-stranded reeler1 RNA in vivo led to reduced nodule formation in bacteria-injected larvae, while the injection of recombinant Reeler1 promoted nodule formation in reeler1 gene-silenced larvae, indicating that Reeler1 is involved in the nodulation response. Knockdown of the reeler1 gene significantly decreased phenoloxidase activity in bacteria-challenged larval hemolymph, while injection of recombinant Reeler1 enhanced phenoloxidase activity, suggesting that Reeler1 is involved in the prophenoloxidase activation cascade. Our results provide new mechanistic evidence about the melanization cascade in the insect immunity.     

感染球孢白僵菌的家蚕免疫应答差异表达基因分析

【目的】筛选家蚕Bombyx mori应对白僵菌Beauveria bassiana侵染的应答基因, 以进一步研究家蚕抵御真菌侵染的分子机制。【方法】采用新一代Solexa高通量测序技术对感染白僵菌及未感染白僵菌的对照组家蚕进行了测序分析, 筛选差异表达基因; 结合生物信息学工具分析差异表达基因的功能注释、 分类及涉及的信号通路等; 应用荧光定量PCR技术验证10个基因的差异表达。【结果】通过测序和生物信息学分析共获得377个差异表达基因, 其中表达上调基因236个, 下调基因141个; KEGG通路分析表明, 各通路中既有表达上调的基因, 也有下调基因; 12个上调基因、 26个下调基因参与3个显著性富集的KEGG通路, 即核糖体、 氨酰tRNA生物合成和剪接体通路。定量PCR与测序结果显示, 溶菌酶、 热激蛋白、 谷胱甘肽S-转移酶、 肽聚糖识别蛋白等与免疫应激相关的蛋白基因均呈现表达上调。【结论】本研究筛选获得的差异表达基因, 特别是上调表达的基因可能与家蚕应对白僵菌侵染的应答机制有关, 其中与免疫应激相关的蛋白基因如溶菌酶、 热激蛋白、 谷胱甘肽S 转移酶、 肽聚糖识别蛋白基因等可能直接参与了家蚕对白僵菌的免疫识别和防御, 研究结果为从分子水平阐明家蚕抵御真菌侵染的防御机制和白僵菌对家蚕的致病机理提供新的依据。     

Temporal organization of endocrine events in relation to the circadian clock during larval-pupal development in Samia cynthia ricini

The temporal organization of secretion of the prothoracicotropic hormone (PTTH) and ecdysone during larval-pupal development of Samia cynthia ricini was studied by ligations, with particular attention to the circadian control of the timing of hormone release. PTTH and ecdysone are required first for the induction of prodromes of pupation and again later for pupal-cuticle formation. PTTH release in the first step occurs during the second or third photophase after the last-larval ecdysis under a photoperiod of 12 hr light and 12 hr darkness and is thought to be under the control of a circadian clock. Ecdysone release follows 1.5 days later, i.e. during the scotophase that precedes the gut purge. In the second secretory step, PTTH is released 2 days after purging the gut, and ecdysone release follows 6 hr later. The PTTH release at this time occurs at a fixed time after the gut purge irrespective of light conditions, accounting for light insensitivity of the timing for pupal ecdysis. Possible mechanisms relating to the inconsistent association of a circadian clock with PTTH release, and those underlying the determination of timing of the gut purge are discussed.     

桑叶营养对家蚕血淋巴阳离子水平的调节作用

调查了取食不同品种桑树（M-5, S-36和V-1）叶片的家蚕（多化性品种Pure Mysore、 二化性品种NB4D2和CSR2）5龄幼虫血淋巴中阳离子的变化。结果表明: 家蚕幼虫血淋巴中Na+浓度低, K+和Mg2+浓度很高, Ca2+浓度较高。在幼虫活跃取食期间, 血淋巴中阳离子水平显著提高。血淋巴中阳离子水平与排泄物的量呈负相关。血淋巴中阳离子水平受阳离子外泌的控制。与非取食阶段相比, 取食阶段的阳离子清除速率低。家蚕二化性品种的血淋巴阳离子水平比多化性品种PM高出30%。结果说明家蚕品种对家蚕血淋巴阳离子变化的影响显著大于桑树品种或家蚕个体发育的影响。